foods
Article
Assessment of the Antibiotic Resistance Profile,
Genetic Heterogeneity and Biofilm Production of
Methicillin-Resistant Staphylococcus aureus (MRSA)
Isolated from The Italian Swine Production Chain
Pedro Rodríguez-López 1 , Virginia Filipello 2 , Pierluigi Aldo Di Ciccio 3 , Alessandra Pitozzi 2 ,
Sergio Ghidini 1 , Federico Scali 2 , Adriana Ianieri 1 , Emanuela Zanardi 1 , Marina Nadia Losio 2 ,
Ancuta Cezara Simon 1, * and Giovanni Loris Alborali 2
1
2
3
*
Department of Food and Drug, Università di Parma, Strada del Taglio 10, 43126 Parma, Italy;
pedro.rodriguezlopez@unipr.it (P.R.-L.); sergio.ghidini@unipr.it (S.G.); adriana.ianieri@unipr.it (A.I.);
emanuela.zanardi@unipr.it (E.Z.)
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna—Headquarters, Via A. Bianchi,
9, 25124 Brescia, Italy; virginia.filipello@izsler.it (V.F.); alessandra.pitozzi@izsler.it (A.P.);
federico.scali@izsler.it (F.S.); marinanadia.losio@izsler.it (M.N.L.); giovanni.alborali@izsler.it (G.L.A.)
Dipartimento di Scienze Veterinarie, University of Turin, Largo P. Braccini 2, Grugliasco, 10095 Turin, Italy;
pierluigialdo.diciccio@unito.it
Correspondence: ancutacezara.simon@unipr.it
Received: 29 July 2020; Accepted: 15 August 2020; Published: 19 August 2020
Abstract: The main aim of the present study was to evaluate the level of antibiotic resistance,
prevalence and virulence features of methicillin-resistant Staphylococcus aureus (MRSA) isolated from
heavy swine at abattoir level and farming environments in Lombardy (Northern Italy). With this
scope, 88 different heavy swine farms were surveyed, obtaining a total of n = 440 animal swabs and
n = 150 environmental swabs. A total of n = 87 MRSA isolates were obtained, with an overall MRSA
incidence of 17.50% (n = 77) among animal samples and a 6.67% (n = 10) among environmental.
Molecular characterisation using multilocus sequence typing (MLST) plus spa-typing showed that
sequence type ST398/t899 and ST398/t011 were the most commonly isolated genotypes, although other
relevant sequence types such as ST1 or ST97 were also found. A lack of susceptibility to penicillins,
tetracycline and ceftiofur was detected in >91.95, 85.05 and 48.28% of the isolates, respectively.
Resistance to doxycycline (32.18%), enrofloxacin (27.59%) and gentamicin (25.29%) was also observed.
Additionally, a remarkable level of antibiotic multiresistance (AMR) was observed representing a
77.01% (n = 67) of the obtained isolates. Genetic analysis revealed that 97.70% and 77.01% of the
isolates harboured at least one antibiotic resistance or enterotoxin gene, respectively, pointing out a
high isolate virulence potential. Lastly, 55.17% (n = 48) were able to produce measurable amounts of
biofilm after 24 h. In spite of the current programmes for antibiotic reduction in intensively farming,
a still on-going high level of AMR and virulence potential in MRSA was demonstrated, making
this pathogen a serious risk in swine production chain, highlighting once more the need to develop
efficient, pathogen-specific control strategies.
Keywords: antibiotic susceptibility; antibiotic resistance; food safety; food hygiene; MRSA; prevalence;
public health; Staphylococcus aureus; survey
1. Introduction
Currently, the emergence of antimicrobial-resistance (AR) in bacteria represents a major challenge
for public health. Poor biosecurity practises and the use, misuse and overuse of antimicrobial
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agents as therapeutic drugs in human medicine, animal farming and in agricultural settings may
apply an environmental pressure favouring the selection of AR bacteria [1]. In addition to this fact,
the inappropriate use of biocides in farms and food industries may select for a subset of resistant
population or even induce resistance to unrelated and clinically relevant antibiotics, in a phenomenon
known as antimicrobial cross-resistance [2–4]. To date, the contribution of the contaminated
environment to the spread of antimicrobial-resistant microorganisms is not fully understood. In this
context, a “one health” approach with an integrated multi-sectorial effort involving animals and their
surrounding environment is needed to tackle the spread of AR in the food chain. In detail, the presence
of bacterial biofilms can also promote the emergence of AR by spontaneous mutation of bacteria or by
means of plasmid-mediated horizontal gene transfer [5].
Methicillin-resistant Staphylococcus aureus (MRSA) is the most commonly identified
antibiotic-resistant pathogen worldwide [6]. It is resistant to almost all β-lactams and can also show
resistance to other major antimicrobial classes such as fluoroquinolones [3,7]. The livestock-associated
MRSA (LA-MRSA) is considered a serious concern for the risks of zoonotic transmission, not only
to people with occupational livestock exposure [8,9], but also to the community through the food
chain [10]. The foremost common LA-MRSA worldwide is the sequence type (ST) 398 belonging to
clonal complex (CC) 398 and the main reservoir for this clone are pigs [11,12]. After its first isolation
from pigs in France [13], ST398 was increasingly detected throughout Europe. MRSA infection or
colonisation in pigs may results in economic losses in the food industry and could represent a zoonotic
risk for humans in contact with infected or colonised animals [14–16].
Even though MRSA transmission is mainly due to human-to-human contact, several studies have
demonstrated that the animal–human transmission (i.e., at abattoir level) cannot be neglected due to
its implications in MRSA spreading [17–19]. Italian swine farming is mainly aimed at the production
of heavy swine for cured meat production, slaughtered at 165–170 Kg live weight i.e., approximately
9 months of life. The major pig production basin is located in three regions of Northern Italy i.e.,
Emilia Romagna, Lombardy and Piedmont accounting for 78.59% of Italian pigs according to 2019
data [20]. Monitoring of MRSA strains in Italian pig production chain is thus of upmost importance
to obtain early indication data of local prevalence and to evaluate changes in AMR and to diminish
its dissemination.
Some studies reported a high prevalence (34.9–38.1%) of LA-MRSA from pig holdings in
Italy [21,22]. Furthermore, an estimated MRSA prevalence of 37.6% was recently reported in
slaughtered pigs of two industrial abattoirs in Southern Italy [23]. The presence of biofilm in
farm or food processing environment could play an important role in MRSA spreading throughout
the food chain [24]. These structures are characterised by an enhanced capacity to endure harsh
environmental conditions such as cleaning and disinfection protocols commonly used in food-related
premises and therefore acting as a pathogen reservoir [25–27]. With this regard, the study carried out
by Vergara et al. [28] demonstrated that MRSA strains isolated from the food sector can form biofilm on
various food-related surfaces when they are exposed to conditions simulating those in food processing
plants. Additionally, the biofilm-forming ability of MRSA that are potentially enterotoxin producers
should be of concern for food safety [28]. Currently, research studies on the AMR and ability of biofilm
formation of MRSA are more common in strains coming from clinical scenarios when compared to
those involving strains isolated from food-related environments. Consequently, it is necessary to also
monitor the prevalence and biofilm formation capacity of MRSA present in the food supply chain.
Considering the great concern of the presence and dispersion of LA-MRSA clones in
food-producing animals such as pigs, which have a zoonotic potential, and their high isolation rate from
Italian intensive pig farms, the aims of this survey were: (i) to determine the antimicrobial-resistance
and heterogeneity of MRSA found in Italian heavy swine production chain, (ii) to analyse the population
structure of these MRSA strains and (iii) to provide data on biofilm formation of the obtained isolates.
Foods 2020, 9, 1141
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2. Materials and Methods
2.1. Sampling Design
A survey was carried out in a total of 88 heavy pig (Sus scrofa domesticus) farms (industrial livestock
farming) located in Lombardy (Northern Italy) between 2016 and 2018. Overall, these farms represent
about 20% of the total Italian heavy swine production and slaughtered animals (19.93% in 2018 and
19.75% according to latest Eurostat data [29]). From each farm, 5 different animals were randomly
selected and animal samples were collected at abattoir level after stunning by rubbing a neck area of
50 × 50 cm using a sterile cotton swab, 1 per animal, thus obtaining a total number of n = 440 samples.
In addition to this, in 50 pig farms, different from those previously sampled, environmental
samples were collected on a voluntary basis. Specifically, sterile cotton swabs were used to pick up
samples from 3 different locations, i.e., barn, aeration device and feeder, obtaining a total number of
samples of n = 150.
In all cases, swabs were then stored at 4 ◦ C and processed within 6 h after sampling.
2.2. Isolation of Suspected MRSA
Samples were enriched in brain hearth infusion (BHI, Oxoid, Hampshire, England) containing
6.5% NaCl (Biolife, Milan, Italy) and incubated for 18–24 h at 37 ◦ C. Next, 0.1 mL of the resulting broth
was plated onto CHROMID® MRSA plates (bioMérieux, Marcy-l’Étoile, France) and Baird-Parker agar
supplemented with rabbit plasma fibrinogen (BP-RPF; Oxoid) following the ISO 6888-2 norm [30].
Suspected colonies were further subjected to Gram staining, coagulase and urease test. Finally, for each
sample, one suspected colony was struck onto tryptone soy agar (TSA, Oxoid) and incubated at 37 ◦ C
for 24 h for further analysis.
2.3. MRSA Confirmation
Confirmation of suspected colonies was performed via detection of nuc and mecA genes using a
duplex colony PCR following the protocol reported by Pichon et al. [31]. Amplicons were resolved in a
1.5% agarose gel (Agarose Multi-Purpose, Roche) stained with GelRed (Biotium, Hayward, CA, USA)
using 1 X Tris-Acetate-EDTA (TAE) buffer (Merck KGaA, Darmstadt, Germany). GeneRuler 100 bp
Plus DNA Ladder (ThermoFisher Scientific, Vilnius, Lithuania) was used as a molecular marker and
finally visualisation was carried out under UV light.
In the case of positive PCR confirmation, working cultures were prepared by mixing a loopful
from TSA plates into a 2 mL sterile cryogenic vial containing 1 mL of sterile 50% glycerol. Cultures
were stored at −20 ◦ C for further analysis.
2.4. Antibiotic Resistance Profiling of Confirmed MRSA Isolates
2.4.1. Disk Diffusion Assays
Isolates identified as MRSA were tested for susceptibility to a panel of 14 antimicrobial agents
using the disk diffusion method following the Clinical & Laboratory Standards Institute indications [32].
The antibiotic disks (Liofilchem) were as follows: amoxicillin/clavulanic acid (AMC; 30 µg),
ampicillin (AMP; 10 µg), ceftiofur (FUR; 30 µg), cephalothin (KF; 30 µg), chloramphenicol (C; 30 µg),
doxycycline (DXT; 30 µg), enrofloxacin (ENR; 5 µg), florfenicol (FFC; 30 µg), gentamicin (CN; 10 µg),
oxacillin (OX; 1 µg), penicillin (P; 30 µg), trimethoprim/sulfamethoxazole (SxT; 25 µg), tetracycline
(TET; 30 µg) and tiamulin (T; 30 µg). A maximum of 6 disks were used in each plate and were incubated
at 37 ◦ C for 24 h. After incubation, inhibition zones were measured with a calliper and isolates were
classified as resistant or non-resistant. Of note, those classified as intermediate were included in the
latter category. Isolates were considered as antibiotic-multiresistant (AMR) if presented resistance to
three or more antibiotic categories [33].
Foods 2020, 9, 1141
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2.4.2. Minimum Inhibitory Concentration Assays
In those determined as AMR isolates, the minimum inhibitory concentrations (MIC) to those
antibiotics considered as critically important for both veterinary and human medicine [34,35]
were determined using the following MIC test strips (Liofilchem): AMC (0.016–256 mg/L),
CN (0.064–1024 mg/L), DXT (0.016–256 mg/L), ENR (0.002–32 mg/L), FFC (0.016–245 mg/L),
SxT (0.002–32 mg/L), TET (0.016–245 mg/L). Plates were incubated at 37 ◦ C, 24 h and results were
compared using available data at the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) [36].
2.4.3. Antimicrobial Resistance Genes Determination
Five different in-house customised PCR protocols were used to determine the presence of genes
involved in antimicrobial resistance using primers targeting for genes related to resistance to β-lactams
(blaZ [17]), tetracyclines (tetL, tetM [17]), quinolones (norA [37]), amphenicols (cfr [38], fexA [39]),
aminoglycosides (aacA-aphD, aadD [17]), pleuromutilins (vgaA [40] and vgaE [41]), trimethoprim (dfrA,
dfrD, dfrK [17]) and macrolides (ermT [17]). A complete list of primer names, sequences, expected
product and references is provided in Table S1. In all cases, extraction of total DNA and amplicon
resolving was performed as described in Section 2.3. PCR reactions were carried out in a final volume
of 15 µL containing 1X Hot Star Master Mix (Qiagen, Hilden, Germany), 0.5 µM of each primer and
3 µL of template DNA. PCR conditions were as follows:
•
•
•
•
•
Multiplex 1 (aacA-aphD, tetL, tetM, dfrA, dfrD): 95 ◦ C (5 min), followed by 32 cycles of 95 ◦ C (30 s),
45 ◦ C (45 s), 72 ◦ C (1 min) and a final extension at 72 ◦ C (10 min).
Multiplex 2 (blaZ, dfrK, vgaE): 95 ◦ C (5 min), followed by 32 cycles of 95 ◦ C (30 s), 47 ◦ C (45 s),
72 ◦ C (50 s) and a final extension at 72 ◦ C (10 min).
Multiplex 3 (ermT, norA, vgaA): 95 ◦ C (5 min), followed by 31 cycles of 95 ◦ C (30 s), 49 ◦ C (40 s),
72 ◦ C (1 min) and a final extension at 72 ◦ C (10 min).
Multiplex 4 (aadD, cfr): 95 ◦ C (5 min), followed by 31 cycles of 95 ◦ C (30 s), 52 ◦ C (40 s), 72 ◦ C (50 s)
and a final extension at 72 ◦ C (10 min).
PCR 5 (fexA): 95 ◦ C (5 min), followed by 31 cycles of 95 ◦ C (30 s), 56 ◦ C (40 s), 72 ◦ C (1 min) and a
final extension at 72 ◦ C (10 min).
2.5. Enterotoxin Gene Determination
The presence of genes related with staphylococcal enterotoxins A (sea), B (seb), C (sec), D (sed)
and E (see) [42] was determined using the primer sets listed in Table S1. A multiplex PCR protocol
was followed as described by Bianchi et al. [43]. In all cases, PCR reaction products were resolved as
described in Section 2.3.
2.6. MRSA Molecular Subtyping and Phylogenetic Analysis
Samples identified as MRSA were subtyped with multilocus sequence typing (MLST) as described
by Enright et al. [44]. To determine the sequence types (STs), data available on the Staphylococcus aureus
MLST database [45] were used. For the spa typing, the spa gene of MRSA strains was amplified by PCR
as described by Shopsin et al. [46], and spa subtypes were determined with Ridom StaphType software
(Ridom GmbH Würzburg, Münster, Germany). A 3500xL genetic analyser (Applied Biosystems, Foster
City, CA, USA) was used to obtain all the DNA sequences. Following this, phylogenetic analysis was
conducted following the protocol described previously by Filipello et al. [19].
2.7. Quantification of Biofilm Production
The ability of MRSA isolates to produce biofilms on flat-bottomed polystyrene microplates was
determined according to the protocol described by Stepanović et al. [47] with slight modifications.
In all cases, all experiments were repeated in triplicate.
Foods 2020, 9, 1141
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First, reactivation of the cells was carried out by transferring 100 µL of working cultures into 5 mL
of TSB and incubated overnight at 37 ◦ C. Next, cultures were adjusted to Abs700 = 0.1 ± 0.001 in sterile
saline using UV visible spectrophotometer (Varian SII Scan Cary 100 Spectrophotometer—Agilent
Technologies, Santa Clara, CA, USA), corresponding to a concentration about 108 CFU/mL, according
to previous calibrations. Adjusted cultures were 1:100 diluted in sterile fresh TSB + 1% glucose (Merck,
Darmsatdt, Germany) reaching a final concentration of about 106 CFU/mL. In all cases, inocula cellular
density was checked by plating onto TSA.
Following this, 200 µL of inoculum was transferred to seven wells of a sterile 96-well flat-bottomed
polystyrene plate (ThermoFisher Scientific). In all cases 200 µL of sterile TSB + 1% glucose was used as
negative control. Then plates were covered and incubated for 24 h at 37 ◦ C in static conditions. After
incubation, bulk culture was pipetted out, and each well was carefully washed three times with 300 µL
of sterile phosphate-buffered saline (PBS, Oxoid) in order to remove loosely attached cells. Samples
were then fixed at 60 ◦ C for 1 h and stained with 150 µL of 2% crystal violet solution (CV; Condalab,
Madrid, Spain) for 15 min. Excess staining was removed by washing wells with running tap water and
allowed to air dry. Finally, 150 µL of 95% ethanol was added to each well to de-stain the wells and let
to dwell for 30 min at room temperature.
Quantitative analysis was performed by measuring the absorbance at 540 nm using a microplate
reader (Microscan FC, Thermo Scientific, Boston, MA, USA). All results were expressed by calculating
the biofilm production index (BPI) as follows: OD (OD mean sample–OD negative control), whereas
OD for the negative control was measured as ODnc : OD negative control + (3 × standard deviation
of the negative control). Strains showing ability to produce biofilm were classified as weak
(ODnc < OD ≤ 2 × ODnc ), moderate (2 × ODn c < OD ≤ 4 × ODnc ) or strong (OD > 4 × ODnc )
biofilm producers [47].
2.8. Statistical Analysis
A two-sided chi-square (X2 ) test using IBM SPSS Statistics for Windows, Version 23.0 (IBM Corp.
Armonk, NY, USA) was conducted to determine the correlation between the biofilm formation ability
and the antibiotic multiresistance of the MRSA isolates. A p-value of <0.05 was deemed to be significant.
3. Results
3.1. MRSA Detection
A total of 440 animal samples (i.e., swabs) obtained from 88 different fattening units at abattoir
level were screened in order to determine the presence of MRSA by means of enrichment culture
and isolation using agar plating with PCR confirmation of mecA gene. Overall, 42 (47.72%) resulted
positive for MRSA. In samples from animals after stunning, positivity was detected in 17.50% (n = 77)
of the analysed samples
Regarding environmental samples (n = 150) from 50 different fattening units, positivity was found
in 14% (n = 7) of the units surveyed. Molecular confirmation via mecA detection gave MRSA positivity
in 6.67% (n = 10) of the environmental samples collected. Consequently, the overall incidence of
MRSA-positive samples was 14.75% (n = 87).
3.2. Antibiotic Resistance Profiles of MRSA Isolates
3.2.1. Phenotypic Resistance
High resistance in β-lactams, especially penicillins (>91% of the isolates), 3rd generation
cephalosporin (i.e., ceftiofur, 48.28%), and tetracycline (85.05%), was observed in MRSA isolates
(Figure 1). With regard to the rest of the antibiotics tested, percentage of resistance among MRSA
isolates ranged between 36.78% (n = 32) in tiamulin (pleuromutilin) and 10.34% (n = 9) in the case
of trimethoprim/sulfamethoxazole (Figure 1). Of note, almost all MRSA isolates (85 out of 87) tested
Foods 2020, 9, 1141
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displayed resistance to at least three different antibiotics. The only two exceptions were isolate SA14
exhibiting only resistance to oxacillin and isolate SA4 with no phenotypic antibiotic resistance detected
(Table S2).
Antibiotic multiresistance (AMR), i.e., resistance to three or more classes of antibiotics [33],
was observed in 77.01% (n = 67) of the MRSA isolates obtained. Taking a closer look, some of the
antibiotic patterns were repeated among the isolates. Specifically, a 62.69% (n = 42) of the isolates
presented an antibiotic resistance profile that was present in at least two of the isolates (Table 1).
It is important to highlight that these observed patterns were not exclusively associated to a given
genotype (i.e., ST/spa-type) but a genetic heterogeneity was observed (Table 1). Of note, among those
that belonged to the same genotype, differences either in the antimicrobial genes, in the enterotoxin
genes or both were detected, indicating that even though closely related, the isolates were different to
one another (results not shown).
Table 1. Major antimicrobial patterns and genotypes detected in multiresistant MRSA isolates.
Antibiotic Resistance Pattern
Genotypes (No. Isolates)
P-AMC-OX-AMP-TET-T
P-AMC-OX-AMP-FUR-TET-T
P-AMC-OX-AMP-FUR-TET-ENR
P-AMC-OX-AMP-FUR-TET-DXT-T
P-AMC-OX-AMP-FUR-KF-ENR-CN
P-AMC-OX-AMP-FUR-TET-DXT-ENR
P-AMC-OX-AMP-KF-ENR-CN
P-AMC-OX-AMP-FUR-TET-DXT-C-T
P-AMC-OX-AMP-FUR-TET-C-FFC-CN-SxT
P-AMC-OX-AMP-FUR-TET-DXT-C-FFC-SxT
P-AMC-OX-AMP-FUR-TET-DXT-C-FFC-T-SxT
ST398/t011 (5); ST398/t899 (1); ST398/t4474 (1)
ST398/t011 (5)
ST398/t899 (8); ST97/t4795 (1)
ST398/t011 (6)
ST1/t127 (3)
ST5422/t1730 (1); ST398/t1939 (1)
ST1/t127 (2)
ST398/t011 (2)
ST398/t899 (2)
ST398/t899 (2)
ST398/t899 (2)
P: penicillin; AMP: ampicillin; AMC: amoxicillin/clavulanic acid; OX: oxacillin; KF: cephalothin; FUR: ceftiofur;
TET: tetracycline; DXT: doxycycline; ENR: enrofloxacin; C: chloramphenicol; FFC: florfenicol; CN: gentamycin; T:
tiamulin; SxT: sulfamethoxazole/trimethoprim.
Maximum level of AMR, i.e., lack of susceptibility upon 12 of the 15 molecules tested, representing
six different classes, was displayed by isolates SA3 (P-AMC-OX-AMP-FUR-TE-DXT-C-FFC-CN-T-SxT)
and SA15 (P-AMC-OX-AMP-FUR-TE-DXT-ENR-C-FFC-CN-T). Of note, both were isolated from
environmental sources (Table 2, Table S2).
3.2.2. Minimum Inhibitory Concentration (MIC) Assays
Determination of MIC values in AMR isolates to antibiotics, considered as highly important or
critical for antimicrobial chemotherapy in human (CN), veterinary (DXT, TET) or both (AMC, ENR, SxT,
FFC), was determined in multiresistant isolates by means of miniaturised systems (MIC strips). Results
showed the highest MIC values in TET (MICTET ≥ 6 mg/L), followed by DXT (MICDXT ≥ 8 mg/L), SxT
(MICSxT ≥ 4 mg/L) and CN (MICCN > 1.5 mg/L), which are all above the available EUCAST resistance
breakpoint values [36]. The range of MIC values for those antibiotics in which EUCAST does not
provide a specific breakpoint value were as follows: MICAMC = 0.19–24 mg/L; MICENR = 0.2–>32 mg/L;
MICFFC = 3–>256 mg/L).
3.2.3. Presence of Antibiotic Resistance Genes
Results obtained demonstrated that all MRSA, except isolate SA6, turned out to be positive in
PCR assays and carried at least one antibiotic resistance gene (Table 2). Specifically, 82.75% (n = 72)
of the MRSA isolates carried dfrA, followed by tetM (81.61%, n = 71), blaZ (78.16%, n = 68) and norA
(77.01%, n = 67). In a lesser extent, dfrD, ermT and cfr genes were detected in 73.79 (n = 12), 12.64
(n = 11) and 8.05% (n = 7), respectively. As displayed in Table 2, a high variability in the combination
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of genes related with antibiotic resistance was observed, with blaZ, tetM, norA, vgaE, dfrA, and dfrK
being the most detected pattern in 10.35% (n = 9) of the isolated MRSA.
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Table 2. Complete list of codes, sources, molecular characterisation and quantification of biofilm production of confirmed MRSA isolates obtained in this study.
Isolate ID
Source
ST
SA1
SA2
SA3
SA4
SA5
SA6
SA7
SA8
SA9
SA10
SA11
SA12
SA13
SA14
SA15
SA16
SA17
SA18
SA19
SA20
SA21
SA22
SA23
SA24
SA25
SA26
SA27
SA28
SA29
SA30
SA31
SA32
SA33
Animal
Environment
Environment
Environment
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Environment
Environment
Environment
Animal
Environment
Environment
Environment
Environment
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
398
398
398
398
97
97
5422
398
398
97
398
398
398
398
30
398
398
398
398
398
398
398
398
4894
398
398
398
398
398
398
398
97
398
spa-Type
t011
t899
t899
t899
t4795
t4795
t1730
t899
t011
t1730
t899
t011
t1939
t011
t318
t899
t1939
t034
t18494
t1200
t1200
t18494
t011
t18494
t18494
t899
t899
t899
t899
t899
t899
t1730
t011
Antibiotic Resistance Genes
Enterotoxin Genes
Biofilm Production
blaZ, tetL, tetM, norA, aadD, dfrA, dfrK
blaZ, tetL, tetM, norA, fexA, aacAaphD, aadD, vgaA, dfrA
blaZ, tetL, tetM, norA, fexA, aadD, vgaA, dfrA
tetL, aadD, vgaA
blaZ, tetL, tetM, norA, aadD, vgaA, dfrA
n.d.
blaZ, tetM, norA, dfrA,
tetM, vgaA, dfrA, dfrK
blaZ, tetM, vgaA, dfrA, dfrD
blaZ, tetL, tetM, norA, cfr, dfrA, dfrD
blaZ, tetL, tetM, fexA, aadD, vgaA, dfrA
blaZ, tetL, tetM
blaZ, tetM, norA, dfrA
tetL
blaZ, tetL, tetM, fexA, cfr, aadD, dfrA
blaZ, dfrA
blaZ, tetM, norA, fexA
blaZ, tetL, tetM, norA, fexA, cfr, aadD, dfrA
blaZ, tetM, norA, fexA, dfrA
tetL, fexA, cfr, aadD, dfrK
blaZ, tetM, norA, vgaE, dfrA, dfrK
blaZ, tetL, tetM, vgaE, dfrA, dfrK
tetL, tetM, aadD, vgaE, dfrA, dfrK
cfr
blaZ, tetL, tetM, cfr, dfrD
blaZ, tetM, norA, vgaE, dfrA, dfrK
blaZ, tetM, norA, vgaE, dfrA, dfrK
blaZ, tetM, norA, vgaE, dfrA, dfrK
blaZ, tetM, norA, vgaE, dfrA, dfrK
blaZ, tetM, norA, aadD, vgaE, dfrA, dfrK
tetM, aadD, dfrA
blaZ, tetL, tetM, norA, aacAaphD, aadD, vgaA, vgaE, dfrA, dfrK, ermT
blaZ, tetM, norA, vgaE, dfrA, dfrK
n.d.
seb, sed, see
sea, seb, see
seb, see
seb, see
n.d.
sea, seb, sed
n.d.
seb
sea
sea, seb
sea, seb
sec
sea, seb
sea, seb
see
n.d.
seb, see
sea, seb, see
n.d
seb, sec
n.d.
sea, seb
sea, sec, see
sea, seb, sec
see
sec, see
sec
n.d.
n.d.
see
n.d.
n.d.
Moderate
Strong
Weak
Weak
Moderate
Moderate
Strong
Strong
Moderate
Strong
Weak
Moderate
Weak
Non-producer
Strong
Moderate
Non-producing
Strong
Weak
Moderate
Weak
Weak
Weak
Weak
Weak
Weak
Weak
Weak
Moderate
Weak
Non-producer
Weak
Weak
Foods 2020, 9, 1141
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Table 2. Cont.
Isolate ID
Source
ST
SA34
SA35
SA36
SA37
SA38
SA39
SA40
SA41
SA42
SA43
SA44
SA45
SA46
SA47
SA48
SA49
SA50
SA51
SA52
SA53
SA54
SA55
SA56
SA57
SA58
SA59
SA60
SA61
SA62
SA63
SA64
SA65
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
398
398
398
398
398
398
398
1
1
1
1
1
398
398
398
398
398
398
398
398
398
398
398
398
398
398
398
398
398
398
398
398
spa-Type
t011
t899
t899
t011
t011
t011
t011
t127
t127
t127
t127
t127
t899
t1939
t899
t899
t011
t899
t899
t899
t011
t011
t011
t011
t4474
t4474
t011
t011
t899
t899
t899
t899
Antibiotic Resistance Genes
Enterotoxin Genes
Biofilm Production
blaZ, tetM, norA, aadD, dfrA, dfrK
blaZ, tetM, norA, vgaA, vgaE, dfrA
blaZ, tetM, norA, aadD, vgaE, dfrA, dfrD, dfrK
blaZ, tetM, norA, aadD, vgaE, dfrA, dfrD, dfrK
blaZ, tetL, tetM, norA, aadD, dfrA
blaZ, tetM, norA, aadD, vgaA, vgaE, dfrA, dfrK
tetL, tetM, norA, aadD, vgaE, dfrK
blaZ, norA, aacAaphD, aadD, vgaE, dfrA, dfrK, ermT
blaZ, norA, aacAaphD, aadD, vgaE, dfrA, ermT
blaZ, norA, aacAaphD, aadD, vgaE, dfrA, ermT
norA, aacAaphD, vgaA, vgaE, dfrA, dfrK, ermT
norA, aacAaphD, vgaA, vgaE, dfrA
blaZ, tetM, norA, vgaE, dfrA, dfrK
blaZ, tetM, fexA, cfr, aadD, vgaA, vgaE, dfrA, dfrK
blaZ, fexA, aadD, vgaA, vgaE, dfrA, dfrK
blaZ, tetL, tetM, norA, aacAaphD, aadD, vgaA, vgaE, dfrA, dfrK
blaZ, tetM, norA, aadD, vgaE, dfrA
blaZ, norA, aadD, vgaE, dfrA, dfrD, dfrK
tetM, norA, aadD, vgaE, dfrA, dfrK
blaZ, tetM, norA, vgaA, vgaE, dfrA, dfrK
blaZ, tetM, norA, vgaA, vgaE, dfrA, dfrK
blaZ, tetM, norA, vgaA, vgaE
blaZ, tetM, norA, vgaA, vgaE, dfrK
blaZ, tetM, norA, aadD, vgaE, ermT
blaZ, tetM, norA, vgaA, vgaE, dfrA
blaZ, tetM, norA, vgaE, dfrA
blaZ, tetM, norA, vgaA, vgaE, dfrA
blaZ, tetM, norA, vgaA, dfrA, dfrK
blaZ, tetM, norA, vgaA, vgaE, dfrA, dfrK
blaZ, tetL, tetM, aacAaphD, aadD, vgaA, vgaE, dfrA, dfrK, ermT
blaZ, tetM, norA, dfrA, dfrK
blaZ, tetL, tetM, aacAaphD, aadD, vgaA, vgaE, dfrA, dfrK, ermT
sec, see
sec, see
sea, seb, see
sea, seb
n.d.
sea, seb, sec, sed
seb
sec
seb, sed
seb, sec
sec
seb, sec, see
seb
seb, sec, see
n.d.
sec, see
seb, see
sea, seb
sec, see
seb, sed, see
see
see
n.d.
seb
sec, see
n.d.
see
seb, sec, see
n.d.
sec
seb, see
n.d.
Weak
Weak
Non-producer
Weak
Non-producer
Weak
Weak
Weak
Moderate
Weak
Weak
Non-producer
Non-producer
Non-producer
Non-producer
Non-producer
Non-producer
Moderate
Non-producer
Non-producer
Non-producer
Non-producer
Non-producer
Non-producer
Non-producer
Weak
Non-producer
Weak
Weak
Moderate
Moderate
Weak
Foods 2020, 9, 1141
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Table 2. Cont.
Isolate ID
Source
ST
SA66
SA67
SA68
SA69
SA70
SA71
SA72
SA73
SA74
SA75
SA76
SA77
SA78
SA79
SA80
SA81
SA82
SA83
SA84
SA85
SA86
SA87
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
Animal
398
398
398
398
1
398
398
398
398
398
398
1
1
398
398
398
398
398
398
398
398
398
spa-Type
t4558
t899
t899
t899
t127
t011
t011
t011
t011
t011
t011
t127
t127
t899
t899
t899
t899
t011
t899
t899
t011
t011
Antibiotic Resistance Genes
Enterotoxin Genes
Biofilm Production
blaZ, tetM, norA, fexA, aacAaphD, vgaA, vgaE, dfrA, dfrK
blaZ, tetM, norA, vgaE, dfrA, dfrK
blaZ, tetM, norA, vgaA, vgaE, dfrA, dfrK
blaZ, tetM, norA, vgaA, vgaE, dfrA, dfrK
blaZ, norA, aacAaphD, vgaA, vgaE, dfrA, dfrK, ermT
blaZ, tetM, norA, aadD, vgaA, vgaE, dfrA, dfrD
tetL, tetM, norA, aadD, vgaA, vgaE, dfrA, dfrK
tetM, norA, aadD, vgaA, vgaE, dfrD, dfrK
tetM, norA, aadD, vgaA, vgaE, dfrA, dfrD, dfrK
tetL, tetM, norA, aadD, vgaA, vgaE, dfrA, dfrK
tetM, norA, aadD, vgaE, dfrA, dfrD, dfrK
blaZ, tetL, norA, aacAaphD, aadD, vgaA, vgaE, dfrA, ermT
blaZ, tetL, norA, aacAaphD, aadD, vgaA, dfrA, dfrK, ermT
tetM, norA, vgaE, dfrA
blaZ, tetM, norA, vgaE, dfr
blaZ, tetM, norA, vgaE, dfr
tetM, norA, vgaE, dfrA
blaZ, tetM, norA, aadD, vgaE, dfrA, dfrD
blaZ, tetM, norA, vgaA, vgaE
blaZ, tetM, aadD, vgaA, vgaE, dfrD
blaZ, tetM, norA, dfrA
blaZ, tetM, norA, vgaE, dfrA
sea, sec, sed, see
sec
n.d.
sec
n.d.
sea
sea
sea
seb, see
sea
n.d.
sea
sea
sea
sea
sea, seb
sea, see
see
see
seb
sea
n.d.
Weak
Weak
Weak
Weak
Weak
Weak
Non-producer
Weak
Non-producer
Weak
Non-producer
Weak
Weak
Non-producer
Non-producer
Non-producer
Non-producer
Non-producer
Non-producer
Weak
Non-producer
Non-producer
n.d.: not detected.
Foods 2020, 9, 1141
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Figure 1. Relative abundances of observed antibiotic resistances in MRSA isolates (n = 87) determined
using the disk diffusion (Kirby–Bauer) assay. In each bar depicting each one of the antibiotics used,
the darker parts represent the percentage of resistant isolates, whereas the lighter parts are indicative
of those classified as non-resistant. Antibiotic classes are represented with different colour bars as
β
follows: blue: β-lactams
(P: penicillin; AMP: ampicillin; AMC: amoxicillin/clavulanic acid; OX: oxacillin;
KF: cephalothin; FUR: ceftiofur); yellow: tetracyclines (TET: tetracycline; DXT: doxycycline); pink:
fluoroquinolones (ENR: enrofloxacin); green: amphenicols (C: chloramphenicol; FFC: florfenicol), grey:
aminoglycosides (CN: gentamycin); brown: pleuromutilins (T: Tiamulin); black and white: folate
inhibitors (SxT: sulfamethoxazole/trimethoprim).
3.3. Presence of Enterotoxin Genes
As shown in Table 2, enterotoxin genes were detected in 77.01% (n = 67). In spite of this high
percentage, in most of the isolates just one gene was detected (34.48%, n = 30) and, overall, no more
than four enterotoxin genes were detected in a single MRSA, isolates SA39 and SA67 being the only
ones (Table 2). Specifically, the percentages of enterotoxin gene detection were as follows: seb_37.93%
(n = 33), see_35.63% (n = 31), sea_31.03% (n = 27), sec_25.29% (n = 22) and sed_6.90% (n = 6).
Of note, all isolates with an absence of enterotoxin genes detected, namely, SA1, SA8, SA17, SA22,
SA29, SA30, SA32, SA33, SA38, SA48, SA56, SA62, SA70 and SA87, were identified as multidrug
resistant MRSA (Table 2, Table S2).
3.4. Molecular Subtyping of MRSA Isolates
MRSA isolates’ subtypes were ascertained by means of multilocus sequence typing (MLST) and
spa-typing the isolates whose combination gave a total of 14 different MRSA genotypes (Table 3).
On one hand, MLST outcomes showed that in most cases, positive isolates belonged to ST398 (82.76%,
n = 72) followed by ST1 (9.20%, n = 8) and ST97 (4.60%, n = 4). Minor STs accounted for 1.15% of the
positive samples (n = 1, each) belonging to ST30, ST4894 and ST5422 (Tables 2 and 3).
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Table 3. Molecular genotypes and frequencies observed among MRSA isolates in this study.
Genotype
Number of Isolates (%)
ST398/t899
ST398/t011
ST398/t1939
ST398/t034
ST398/t18494
ST398/t1200
ST398/t4474
ST398/t4558
ST1/t127
ST97/t1730
ST97/t4795
ST4894/t18494
ST30/t318
ST5422/t1730
33 (37.93%)
27 (31.03%)
3 (3.45%)
1 (1.15%)
3 (3.45%)
2 (2.30%)
2 (2.30%)
1 (1.15%)
8 (9.20%)
2 (2.30%)
2 (2.30%)
1 (1.15%)
1 (1.15%)
1 (1.15%)
On the other hand, a higher diversity of spa types was observed when compared with STs (Tables 2
and 3). Specifically, 12 different types were detected, namely, the major spa-type recovered t899 (37.93%,
n = 33) and t011 (31.03%, n = 27). The rest of spa-types were detected in a much lower proportion and
classified as t127 (9.20%, n = 8), t18494 (4.60%, n = 4), t1730 (3.45%, n = 3), t4474 (2.30%, n = 2), t4795
(2.30%, n = 2), t1200 (2.30%, n = 2), t1939 (1.15%, n = 1), t1939 (1.15%, n = 1) and t4558 (1.15%, n = 1).
The full eBURST algorithm was used to describe the different STs based on their genetic proximity.
As depicted in Figure 2, MRSA isolates were clustered into six different STs and, in particular, ST398
was the most variable, grouping eight different spa-types.
Figure 2. Minimum spanning tree of the 87 MRSA isolates analysed with MLST. The diagram was
constructed by the full eBURST algorithm using the StaphType software v 2.0. Each single sequence
type is depicted by a single circle and the size is proportional to the total number of isolates found
within. Different colours in the circle indicate the different spa that are included in the same ST.
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3.5. Biofilm Production Assays
In order to obtain a better picture of the phenotypic characteristics of the acquired isolates, biofilm
production in polystyrene plates was determined through crystal violet staining [48]. Based on the
outcomes obtained, 34.48% (n = 30) of the isolates were classified as non-biofilm producers, whereas
65.52% (n = 57) of the isolates were able to produce quantifiable amounts of biofilm at 24 h. Specifically,
44.83% (n = 39), were weak producers, 13.79% (n = 12), were moderate producers and 6.90% (n = 6),
and were classified as strong producers (Table 2). The latter group included three environmental
isolates (SA2, SA15 and SA18) and three animal isolates (SA7, SA8 and SA10) (Table 2).
Statistical analysis showed no correlation (p > 0.05) between the biofilm forming capacity
(non-producer, weak, moderate and strong producer) and the antimicrobial multiresistance among the
confirmed MRSA isolates.
4. Discussion
According to the latest report of The European Food Safety Authority (EFSA), MRSA prevalence
in pig herds among European member states varied between 0.4 and 90.4% in 2017 with a slight
reduction in 2018 with prevalence values ranging from 0 to 89.2% [49]. In the present work, 88 different
swine farms at abattoir level were sampled obtaining a total of 440 samples. A total of 47.72% of the
fattening units (n = 42) showed MRSA-positive results. It is important to note that this study was
exclusively focused on heavy swine reared in Lombardy, the Italian region, with the main national pig
production [50].
Similar to the outcomes obtained in the present work, results of positivity among pig farms in
Italy have been also observed by Battisti et al. [22], in which a 38.1% of the sampled pig farms were
positive for MRSA. Contrarily, recent studies performed in Italy showed a slightly higher incidence of
MRSA-positive farms compared to the results obtained here. As a matter of illustration, the study
of Parisi et al. [18] demonstrated that 64.7% of the pig farms turned out to be positive for MRSA.
Similarly, Pirolo et al. [51] observed a positivity of MRSA in 66.8% of the swine farms sampled. These
variations in results can be of a diverse nature, such as the use of different sampling, identification and
characterisation methods that add an extra difficulty for prevalence data comparison [49,52].
Over the last years, the increasing resistance to antimicrobials among MRSA present in swine has
been considered an issue of serious concern [21,53]. Antibiotic misuse/overuse in animal fattening units
has posed an environmental pressure leading to the selection of a subpopulation of MRSA resistant
not only to methicillin, but presenting cross-resistance to several other molecules [2–4,7]. This was
observed by Alba et al. [54] in MRSA isolated from cattle farms in different European countries,
demonstrating not only a high level of AMR, but the carriage of several virulence genes, conferring the
fully invasive potential of MRSA to humans. In swine production chain, Lopes et al. [55] observed an
unusual high level of resistance to clindamycin in the ST398 isolate, establishing a direct link between
the use of antibiotics in a given farming unit and the level of resistance of the MRSA population present.
Moreover, recently published data report a resistance to veterinary relevant antibiotics such as tiamulin
of up to 50% among MRSA isolated from fattening pigs, and up to 100% among those isolated from
pig meat [49]. This fact is in contrast with the obtained results, in which a slightly lower tiamulin level
of resistance was observed (Figure 2).
As displayed by the results, a remarkable level of resistance to TET (Figure 2) and the presence
of TET resistance-associated genes (Table 3), reported to be virtually present in all LA-MRSA [56,57],
was demonstrated. This further corroborated data previously reported by several authors regarding TET
resistance among LA-MRSA from pigs [18,22,23,51,57]. The main reason behind such TET-resistance
levels is the intensive use of this antibiotic in farming [58–60] that may favour a cohort selection of
TET-resistant bacteria; this is significantly notable in ST398 [21,53]. Taking a closer look, most of the
obtained ST398 (CC398) isolates present TET resistance, which is in agreement with the latest EFSA
data that claim that TET resistance is commonly associated with CC398 variants [49]. Nevertheless,
among MRSA populations, a dynamic pattern is expected to be found due to natural evolution of this
Foods 2020, 9, 1141
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microorganism together with continuous exposure to antimicrobials and other environmental insults,
thus modelling the overall phenotypic resistance characteristics of LA-MRSA in Italian abattoirs.
Multidrug resistance (MDR) was detected in 76.62% (n = 59) of the analysed MRSA (Table S2).
The most observed resistance pattern was non-susceptibility to penicillin, amoxicillin/clavulanic
acid, oxacillin, ampicillin, and ceftiofur. These observed high levels of resistance to penicillins and
cephalosporins were in consonance with previously published data pointing out β-lactams resistance
of MRSA in food systems [18,61]. It is important to remark that in this study two isolates, namely, SA63
(ST398/t899) and SA78 (ST1/t127), were found to display no susceptibility to 12 different molecules,
respectively, representing 6 different classes. This is in line with previous results published in which
the same MRSA genotypes isolated from the pork supply chain also displayed high levels of MDR [18].
This level of MDR encountered is of a special relevance when also considering the amount of resistance
genes present among isolates and the fact that some of them are encoded in mobile elements that could
further enhance horizontal and vertical resistance gene transfer in swine-related premises [56].
According to the EFSA report on antimicrobial monitoring on MRSA, the main route of transmission
of LA-MRSA into the population is direct contact with animals, e.g., at abattoir level, and then
dissemination [61]. In line with this, the study carried out by Pirolo et al. [62] involving pig farm
workers demonstrated that 21.6% of the workers were colonised by livestock-associated MRSA
(LA-MRSA) and that this colonisation was significantly higher if direct animal–human contact occurred.
Additionally, Cuny et al. [63] detected a 30% antibiotic multiresistance to penicillins, tetracyclines
and macrolides in ST398 MRSA isolated from humans in contact with farm animals, a fact that poses
a therapeutic challenge if infection occurs. However, contamination routes within pig production
environments still remain obscure considering that clones classified as community-acquired MRSA
(CA-MRSA), such as ST1/t127 and ST30/t318, were also found among the MRSA isolated (Table 3),
which is in agreement with previously reported data [64]. Regarding ST30, is important to remark that
despite being commonly categorised as methicillin-sensitive S. aureus (MSSA) [65], MRSA variants
have been isolated from pigs in Australia [66] and Portugal [67] that indicate not only a worldwide
dissemination of this specific ST among pig herds, but tangible evidence of the dissemination of
methicillin resistance via integration of the staphylococcal cassette chromosome mec (SCCmec) among
sensitive strains [68]. Additionally, ST30 showed a remarkably high level of MDR (Table S2), harboured
several antibiotic resistance and enterotoxin genes, and was identified as a strong biofilm producer
(Table 2), which is highly relevant in the food production chain due to its potential harmfulness and
environmental recalcitrance.
In European animal production systems, most efforts to track down MRSA routes of transmission
have been mainly posed in regard to the swine production chain since it has been demonstrated that
pigs are the major reservoir of several livestock-associated MRSA (LA-MRSA) genotypes such as
ST97 [17,18,23], but especially ST398 [21,69]. Results obtained in this survey demonstrated that most
of the MRSA isolates obtained belonged to the ST398, which is in agreement with the EFSA data and
previous studies performed in Italy that highlight that this ST is the most prevalent LA-MRSA among
pig herds [18,21,23,49]. Additionally, molecular characterisation detected eight different spa-types
within ST398, including t011 and t899 with the latter being the most predominant, which is in line
with previously reported data [18,22,53,69,70]. Of note, ST398/t899 have been reported to be among
the major genotypes isolated in fattening pigs at abattoir level in Finland and Spain [49], indicative of a
wide distribution regardless of the European zone. Additionally, in line with our results, recent data
show that a high prevalence of ST398/t011 in Europe has been reported due to an increase in recent
years among Swiss fattening pig herds [49]. Furthermore, a recent study conducted by Larsen et al. [71]
demonstrated a high prevalence of ST398 in humans regardless of their direct contact with livestock
animals, thus indicating that humans themselves could also act as further reservoirs for this specific
lineage. It is important to highlight that ST398 was also present in 9 out of 10 of the environmental
samples (Table 3).
Foods 2020, 9, 1141
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ST97 was also found among the analysed samples (Table 2). In Italy, ST97 (t1730 and t4795) is
considered to be the most prevalent swine-associated lineage after ST398 [22,72]. This LA-MRSA
is commonly associated with infection in cattle, although spreading among pig herds in intensive
farming have been also described [72]. It has been hypothesised that in pig farming, ST97 can acquire
high levels of antibiotic resistance, as observed in our results (Table 1, Table S2), due to the selective
pressure, and they can then be transmitted to humans, causing severe infections [72]. This capability
of interspecies spreading together with the antimicrobial resistance makes ST97 an issue of serious
concern for public health.
Genotype ST1/t127 was also detected (Figure 2, Table 3), which is in line with previously published
data [21,22,53]. Although firstly categorised as community acquired (CA) MRSA [49,73] responsible
for various infections in human [63,74,75], it has been also isolated from livestock animals [18,22,23].
With this regard, Franco et al. [76] carried out a phylogenetic characterisation of human and pig
MRSA ST1/t127 isolates from Italy, Spain and Demark. The results obtained demonstrated that despite
belonging to the same genotype, the homology between the two groups differed by 25%. Moreover,
it was demonstrated that genotype ST1/t127 displayed a high resistance to major antimicrobials, which
is in accordance with the results obtained in the present study (Table 3).
Staphylococcal enterotoxins (SEs) are considered one of the major virulence factors in S. aureus
in the context of food safety [77]. This highly heat-resistant family of proteins can cause severe food
poisoning outbreaks with symptoms including vomiting, abdominal cramping, and even fatality due
to hypersensitivity induction [78], and their synthesis is dependent on the staphylococcal cellular
density [79]. The latter aspect is considered within the EC Regulation 2073/2005 of microbiological
safety of foodstuffs as a safety criterion in some food products of animal origin [80]. In this study,
the enterotoxigenic potential of MRSA was evaluated, demonstrating that 77.01% of the isolates
possessed at least one gene relate to SE synthesis (Table 3). This is in contrast with a previous study
carried out by Normanno et al. [23], where no MRSA isolated from swine carried any SE gene. Similarly,
Kluytmans [10] reported that most MRSA ST398 seldom carry SE genes, which clearly contrasts with the
outcomes in this work since most of the isolates obtained carried at least one (Table 2). It is important to
remark that among the isolated MRSA, a small proportion lacked on genes while presenting an MDR
phenotype, and one of them, SA8, showed a high capacity of biofilm formation (Table 2). This aspect is
important to take into account and highlights once more that the hazardous potential of MRSA strains
found in swine production chain must be assessed from different angles, as a non-SE producer can
harbour several resistance genes and become a persistent strain forming highly stable biofilms.
In scientific literature, it is widely accepted that S. aureus is able to embed itself in a polysaccharide
matrix and form biofilms that adhere onto a wide variety of food-related surfaces, contributing to
its survival to harsh environmental conditions (i.e., presence of antibiotics) alone or in multispecies
communities [24,81]. The mechanisms beneath such resistance are diverse and lack antimicrobial
diffusion into the biofilm matrix and the presence of recalcitrant cells, such as those belonging to the
viable but not cultivable phenotype [82]. In S. aureus, the ability to form biofilms is directly related to the
presence of virulence factors such as adhesins [83], and this structure contributes to the dissemination
of resistance genes in the cells therein [5]. In this study, 65.52% of the resulting MRSA isolates were
classified as biofilm-producing at 24 h. However, different levels of biofilm production were detected,
in which most of the producers displayed low levels of biofilm production (i.e., weak producers,
44.83%) (see Section 3.4. for details). Similar results were previously reported by Di Ciccio et al. [84] in
S. aureus strains from food and food-related environments. Of note, the quantification and subsequent
classification of the biofilm capacity of the MRSA is only valid in the experimental conditions tested
since, as demonstrated by Zhang et al. [83], biomass production, and subsequent classification,
is directly dependent on the age of the biofilm. Based on our results, there was no correlation between
the MDR phenotype and the capacity of a given MRSA of producing a biofilm. With this regard,
a recent study carried out by Ou et al. [85] shows that there is a certain relationship between the level
of antibiotic resistance and the biofilm forming ability in MRSA isolated from food from animals,
Foods 2020, 9, 1141
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but that the biofilm capacity of them is somehow impaired in those displaying the highest level of
antibiotic resistance. Since MRSA biofilms can become recalcitrant contamination foci and a reservoir
of several resistance genes, information about their adhesion capability is especially relevant to assess
the potential hazard of a given subpopulation of MRSA, especially in those related with the food
production chain.
5. Conclusions
Methicillin-resistant Staphylococcus aureus (MRSA) is among the major pathogens in the pig supply
chain, and it is detected in both live and slaughtered animals as well as in meat products that are
currently considered a potential MRSA transmission vector into the population [86,87]. In the present
work, a high diversity of MRSA genotypes present in Italian pork supply chain was demonstrated.
In addition to this, antimicrobial susceptibility evaluation highlighted high levels β-lactams and
tetracyclines resistances among the isolated bacteria that may indicate an environmental selection of
resistant MRSA variants due to the use of antibiotics in intensive animal farming. Additionally, we
demonstrated capability of these MRSA to form biofilms, which gives these bacteria higher capability
to survive in food-related premises. This could be translated into eventual recalcitrance and subsequent
spreading of some genetic subtypes and antimicrobial resistance genes in the swine supply chain.
Lowering the level of antibiotic usage in order to improve animal and human welfare and thus
avoid the selection process due to environmental pressure and dissemination of antibiotic resistances is
an issue of extreme importance. Due to the high prevalence of some MRSA clones, such as ST398, it is
of paramount importance to intensify specifically control measures regarding MRSA colonisation and
transmission in farms and food-processing environments in order to avoid MRSA direct animal–human
or foodborne spreading, through improvement of biosecurity practises, i.e., personal protection
equipment, process environment and cleaning and disinfection procedures.
Supplementary Materials: The following is available online at http://www.mdpi.com/2304-8158/9/9/1141/s1,
Table S1: List of primer sets used in this study; Table S2: Phenotypic antibiotic resistance pattern and minimum
inhibitory concentrations (MIC) to human therapy and/or veterinary relevant antibiotics tested among isolated
MRSA obtained in this study.
Author Contributions: Conceptualisation, A.I. and G.L.A.; methodology, V.F., M.N.L., A.P. and A.C.S.; validation,
E.Z. and S.G.; formal analysis, P.R.-L., F.S. and A.I.; writing—original draft preparation, P.R.-L., A.I., A.C.S.
and P.A.D.C.; writing—review and editing, P.R.-L., A.I., A.C.S., S.G. and P.A.D.C.; supervision, A.I. and G.L.A.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was financially supported by The Italian Ministry of Health as part of the project
CLASSYFARM (Farm risk categorisation and evaluation of the level of antibiotic-resistance in the swine production
chain. www.classyfarm.it).
Conflicts of Interest: The authors declare no conflict of interest.
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